An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-β, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression (, ,, ,, and increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

Evolution of Bioengineered Lung Models: Recent Advances and Challenges in Tissue Mimicry for Studying the Role of Mechanical Forces in Cell Biology

“…However, even this complex 3D multicell model of the lung can only partly reflect the properties of native lung tissue. For instance, it does not represent an intact epithelial‐endothelial tissue barrier, ALI conditions as encountered in the lung and an intact immune system causing nonphysiological responses . Moreover, these top‐down approaches still require the use of animals or donor organs, while bottom‐up approaches bear the potential of animal free drug/toxicity testing.…”

Perlin Noise Generation of Physiologically Realistic Patterns of Fibrosis

“…Non-invasive imaging techniques remain unable to fully characterise the nature of fibrosis in the lung [8] and particularly the heart [9], resulting in a reliance upon ex vivo studies [10,11]. This has motivated in silico investigations, seeking to explain both the creation of different fibrotic patternings [12] and in the case of cardiac fibrosis, its pro-arrhythmic consequences [13,14,15,16,17].…”

Multiomic and quantitative label-free microscopy-based analysis ofex vivoculture and TGFbeta1 stimulation of human precision-cut lung slices

“…To further investigate the functional consequences of highly upregulated profibrotic response we sought to measure the deposition of fibrillar collagen (tissue phenotypic marker of fibrosis) 3 in ECM of hPCLS. Other studies 10,20 have made attempts to use hPCLS for studying fibrotic signalling, however, a functional readout that confirms excess extracellular matrix deposition has been lacking. Not only in this model system but in other systems as well, only indirect readouts such as ELISA 57 of procollagen peptides or hydroxyproline levels in serum have been used to infer on fibrosis, e.g.…”

“…While it is important to measure regulation of various markers of a pro-fibrotic process to validate induction of pro-fibrotic signalling, to better assess the functional consequences it is necessary to confirm that this signalling translates into higher extracellular deposition of fibrillar collagen. Several recent studies 10,20,21 have shown induction of pro-fibrotic signalling in ex-vivo cultured hPCLS using TGFß1 stimulation 10 or a cocktail of pro-fibrotic factors 20 but a validation of consistent and specific ECM deposition (fibrillar collagen in particular) is still lacking. Next we investigated whether the observed upregulation was translating into deposition of fibrillar collagens in the extracellular matrix.…”

“…PCLS (rat lung and human) came into existence in the early 1990s 17,18 but have only recently been used as a model for evaluating human disease pathophysiology 19 . Several groups have used this model system to recapitulate pro-fibrotic signalling 10,20,21 although the mechanisms at play and the validation of the molecular changes that hPCLS undergo in ex vivo culture are still elusive. These studies do show an induction in fibrotic signalling but a functional readout of enhanced ECM deposition is lacking.…”